F04.43 - Cells and Tissue Engineered Constructs for TEMPs

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1.1 This terminology defines basic terms and presents the relationships of the scientific fields related to microphysiological systems (MPS). Committee F04 has defined these terms for the specific purpose of unifying the language used in standards for MPS.  
1.2 The terms and nomenclature presented in this standard are for the specific purpose of unifying the language used in MPS standards and are not intended for labeling of regulated medical products.  
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.4 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through culture expansion, processing, transfection, or genetic modification.  
4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay.  
4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high amounts of osteogenic medium components can lead to dystrophic, pathologic, or artifactual calcium-based precipitates that do not indicate differentiation of the cells in culture to functional osteoblasts (1).4 For example, when high concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell (2, 3).  
4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation, but it can also be stimulated in other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that described in Practice F2997 for the Quantification of ...
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1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells including mesenchymal stromal cells.  
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.  
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the cells.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This practice describes a cell adhesion method that can be used to provide a detachment percent at a given RCF for cells that have adhered to a substrate, typically for a short time. The information generated by this practice can be used to obtain a semi-quantitative measurement of the adhesion of cells to either an uncoated or pre-coated substrate, when compared to a reference (adherent) cell type on the same substrate. As described in Reyes and Garcia (2003), it is recommended that the 50 % point be used for either ligand concentration or RCF for the most robust measurement of adhesion strength. The adhesion may vary due to changes in the phenotype of the cells or as a result of the specific properties of the surface. The substrate may include tissue culture-treated polystyrene, biomaterials, or bioactive surfaces. If the substrate is a hydrogel, care must be taken to avoid cohesive failure in the hydrogel (that is, detached cells have pulled away fragments of gel). The coating may consist of (but is not limited to) the following: natural or synthetic biomaterials, hydrogels, components of extracellular matrix (ECM), ligands, adhesion or bioactive molecules, genes, or gene products. Cell concentration is also critical, as use of too high a concentration of cells may result in cells detaching as a sheet, rather than as individual cells. This centrifugation approach, once validated, may be applicable for quality control (QC) and product development. However, until the method is correlated to other measures of cell attachment, the current method should be run in parallel with other known measures of cell adhesion.  
4.2 This practice does not cover methods to quantitate changes in gene expression, or changes in biomarkers, as identified by immunostaining. This practice additionally does not cover quantitative image analysis techniques. In some cases, the change in adhesive properties may reflect on the degree of differentiation or de-differentiation of the cells...
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1.1 This practice covers a centrifugation cell adhesion assay that can be used to detect changes in adhesive characteristics of cells with passage or treatments. This approach measures the force required to detach cells from a substrate. Adhesion, among many variables, may vary due to changes in the phenotype of the cells.  
1.2 This practice does not cover methods to verify the uniformity of coating of surfaces, nor does it cover methods for characterizing surfaces.  
1.3 The cells may include adult, progenitor, or stem cells from any species. The types of cells may include chondrocytes, fibroblasts, osteoblast, islet cells, or other relevant adherent cell types.  
1.4 This practice does not cover methods for isolating or harvesting of cells. This practice does not cover methods to quantitate changes in gene expression, or changes in biomarker type or concentration, as identified by immunostaining. Nor does this practice cover quantitative image analysis techniques.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 In-vitro cell proliferation assays are used to screen the capability of cells to proliferate and self-renew within scaffolds for regenerative medicine and tissue-engineering applications. The cell proliferation in vitro, in conjunction with other characteristics of the cells such as gene expression, can be used to determine if the cells have maintained their properties.  
5.2 Cell proliferation may be an important parameter to test as a quality attribute of a cell-scaffold construct. This test helps to assess cell colonization within a scaffold.  
5.3 This method provides a technique for vital assessment and quantification of the fluorescence intensity related to dye metabolism by living and proliferating cells. This method assumes that viable cells will have an active metabolism, which is required to support life-associated cellular processes such as the conversion of nutrient sources into energy and proliferation. There may be cells that are not actively proliferating, yet are still viable within the construct. The methods described within this practice enable nondestructive testing for monitoring the cell proliferation kinetics throughout the culture period by repeated analysis at multiple time points on the same test sample with minimal toxicity. This standard practice is written only for resazurin dye, a non-cytotoxic reagent that should not affect cell viability and proliferation at low concentration. This is a distinct advantage over many other reagents used to measure cell number, such as measurements of the intracellular components (such as DNA, protease, or ATP) which require cell lysis and can therefore only be used for endpoint analysis.  
5.4 Resazurin, which has low fluorescence, may be metabolized by cells into resorufin, which is highly fluorescent. An increase in fluorescence caused by the conversion to resorufin may correlate with increased dehydrogenase activity, which may correlate with an increase in cell number and therefore proliferatio...
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1.1 This practice describes how to conduct a nondestructive proliferation test for mammalian cells based on metabolic activity that can be used to assess the number of viable cells within three-dimensional (3D) scaffolds for regenerative medicine and in tissue-engineered medical products (TEMPs).  
1.2 This practice provides a detailed explanation of the resazurin cell metabolic activity method in terms of reagent concentrations, incubation times, cell culture media composition, calibration curve, controls, assay linearity, and limitations of the assay.  
1.3 This practice describes factors that can interfere with accurate cell proliferation assessment.  
1.4 Since the assay has washing steps, it is limited to assessing cells that are immobilized, such as by adhesion to a culture dish, adhesion to a scaffold, or encapsulation in a hydrogel.  
1.5 The assay is limited to cell types that can metabolize resazurin to provide a signal in the assay.  
1.6 This document does not propose acceptance criteria for a cell-based product based on the application of a cell proliferation test method.  
1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcium deposits or mineralized matrix that form in vitro may be an indicator of differentiation to a functional osteoblast; however, expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast.  
5.2 This practice provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, XO. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to ensure that the images have not been underexposed or overexposed. Intensity and area do not correlate directly to calcium content.  
5.3 XO enables the monitoring of calcium deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of the mineralized area due to dye accumulation from repeated application (1).3 Calcium deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose (1) but require a different procedure for analysis than XO (that is, concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects.  
5.4 The practice may be applied to cultures of any cells capable of producing calcium deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization.  
5.5 During osteoblast differentiation assays, osteogenic supplements are ...
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1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium-chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice.  
1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (that is, osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice.  
1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage (area%), for example, fraction of coverage of the area analyzed.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibili...

SIGNIFICANCE AND USE
4.1 The Manual Observer-Dependent Assay-The manual quantification of cell and CFU cultures based on observer-dependent criteria or judgment is an extremely tedious and time-consuming task and is significantly impacted by user bias. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell- and colony-based assays often require that a single observer count cells and colonies in hundreds, and potentially thousands of cultures. Due to observer fatigue, both accuracy and reproducibility of quantification suffer severely (5). When multiple observers are employed, observer fatigue is reduced, but the accuracy and reproducibility of cell and colony enumeration is still significantly compromised due to observer bias and significant intra- and inter-observer variability (2, 4) . Use of quantitative automated image analysis provides data for both the number of colonies as well as the number of cells in each colony. These data can also be used to calculate mean cells per colony. Traditional methods for quantification of colonies by hand-counting coupled with an assay for cell number (for example, DNA or mitochondrial) remains a viable method that can be used to calculate the mean number of cells per colony. These traditional methods have the advantage that they are currently less labor intensive and less technically demanding (8, 9). However, the traditional assays do not, provide colony level information (for example, variation and skew), nor do they provide a means for excluding cells that are not part of a colony from the calculation of mean colony size. As a result, the measurement of the mean number of cells per colony that is obtained from these alternative methods may differ when substantial numbers of cells in a sample are not associated with colony formation. By employing state-of-the-art image acquisition, processing and analysis hardware and software, an accurate, precise, robust and automated ana...
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1.1 This practice, provided its limitations are understood, describes a procedure for quantitative measurement of the number and biological characteristics of colonies derived from a stem cell or progenitor population using image analysis.  
1.2 This practice is applied in an in vitro laboratory setting.  
1.3 This practice utilizes: (a) standardized protocols for image capture of cells and colonies derived from in vitro processing of a defined population of starting cells in a defined field of view (FOV), and (b) standardized protocols for image processing and analysis.  
1.4 The relevant FOV may be two-dimensional or three-dimensional, depending on the CFU assay system being interrogated.  
1.5 The primary unit to be used in the outcome of analysis is the number of colonies present in the FOV. In addition, the characteristics and sub-classification of individual colonies and cells within the FOV may also be evaluated, based on extant morphological features, distributional properties, or properties elicited using secondary markers (for example, staining or labeling methods).  
1.6 Imaging methods require that images of the relevant FOV be captured at sufficient resolution to enable detection and characterization of individual cells and over a FOV that is sufficient to detect, discriminate between, and characterize colonies as complete objects for assessment.  
1.7 Image processing procedures applicable to two- and three-dimensional data sets are used to identify cells or colonies as discreet objects within the FOV. Imaging methods may be optimized for multiple cell types and cell features using analytical tools for segmentation and clustering to define groups of cells related to each other by proximity or morphology in a manner that is indicative of a shared lineage relationship (that is, clonal expansion of a single founding stem cell or progenitor).  
1.8 The characteristics of individual colony objects (cells ...

SIGNIFICANCE AND USE
4.1 Cell attachment or, lack of it, to biomaterials is a critical factor affecting the performance of a device or implant. Cell attachment is a complicated, time-dependent, process involving significant morphological changes of the cell and deposition of a bed of extracellular matrix. Details of the adhesive bond that is formed have been reviewed by, for example, Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and Garcia and Gallant (2003) (6). The strength of this coupling can be determined either by monitoring the force of attachment between a cell and a substrate over time or by measuring the force required to detach the cell once it has adhered.  
4.2 Cell adhesion to a surface depends on a range of biological and physical factors that include the culture history, the age of the cell, the cell type, and both the chemistry and morphology of the underlying surface and time. These elements need to be considered in developing a test protocol.  
4.3 Devising robust methods for measuring the propensity of cells to attach to different substrates is further complicated since either cell adhesion or detachment can be assessed. These processes are not always similar or complementary.  
4.4 Most studies of cell attachment focus on obtaining some measure of the time-dependent force required to detach, or de-adhere, cells that have already adhered to a surface (James et al, 2005) (7). More recently investigators have begun to measure the adhesive forces that develop between cells and the underlying surface during attachment (Lukas and Dvorak, 2004) (5). From a practical point of view, it is much easier to measure the force required to detach or de-adhere cells from a surface than to measure those that develop during attachment. However, in both cases, the experimental data should be interpreted with a degree of caution that depends on the intended use of the measurements. The methods of measuring cell adhesion described herein are measures of the force required to ...
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1.1 This guide describes protocols that can be used to measure the strength of the adhesive bond that develops between a cell and a surface as well as the force required to detach cells that have adhered to a substrate. Controlling the interactions of mammalian cells with surfaces is fundamental to the development of safe and effective medical products. This guide does not cover methods for characterizing surfaces. The information generated by these methods can be used to obtain quantitative measures of the susceptibility of surfaces to cell attachment as well as measures of the adhesion of cells to a surface. This guide also highlights the importance of cell culture history and influences of cell type.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine in vivo product performance of cell/biomaterial constructs (see 5.7); therefore, there is a need for standardized test methods to quantify cell viability.  
5.2 There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells onto scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell numbers within a given methodology.  
5.3 As described in Guide F2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantified using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.  
5.4 These test methods can be used to quantify cells on non-porous or within porous hard or soft 3-D synthetic or natural-based biomaterials, such as ceramics, polymers, hydrogels, and decellularized extracellular matrices. The test methods also apply to cells seeded on porous coatings.  
5.5 Test methods described in this guide may also be used to distinguish between prolifer...
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1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue-engineered medical products (TEMPs).  
1.2 In addition to providing a compendium of available techniques, this guide describes materials-specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid or account for, or both, scaffold material/cell viability assay interactions.  
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.  
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide describes markers involved in myoblast differentiation that can be used to screen stem cells to help define myogenic capacity. Stem cells include pluripotent and multipotent stem cells capable of differentiating into several different mesenchymal cells, including skeletal muscle myoblasts.  
5.2 To assess myogenesis in cells derived and not derived from muscle, markers are measured to accurately define the changes in transcription and structural proteins that regulate differentiation, fusion, and myotube formation. Discussion of these markers is important to understand why they are recommended.  
5.3 Myogenic Differentiation:  
5.3.1 Myogenic differentiation is a highly regulated process controlled by paired box (Pax) transcription factors and the myogenic regulatory factor (MRF) family. During early differentiation in adults, myogenic progenitors such as activated satellite cells or myoblasts express Pax3 and Pax7. Pax3 and Pax7 transcription factors switch the cells toward a myogenic fate, and repress myocyte differentiation (2), priming the cell for later MRFs. To form muscle, the family of MRFs is required to terminally differentiate myoblasts and form myofibers. These regulatory proteins belong to a superfamily of basic helix-loop-helix transcription factors that consists of myogenic differentiation factor 1 (Myod1), myogenic factor 5 (Myf5), myogenin (Myog), and myogenic factor 6 (Myf6). In the initial stages of myogenic differentiation, Myod1 and Myf5 are the first MRFs to be expressed, and trigger increased production of Myog and Myf6  (3). Increased intracellular Myog and Myf6 induces terminal differentiation of myoblasts into myocytes, leading to fused myotubes.  
5.4 Forming Myotubes:  
5.4.1 While myogenic markers describe differentiation, fusion into multinucleated myotubes is an important factor in muscle biology. Myoblasts differentiate into a fusogenic phenotype characterized by multiple fusion markers. One marker of note is m-c...
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1.1 Myogenic differentiation is a process regulated by specific transcription factors and signaling molecules that have been shown to induce a myogenic phenotype. Transcription factors mark the stages of myogenesis and act as benchmarks for use in myogenic assays.  
1.2 This guide applies to mammalian cells but does not apply to non-mammalian cells as the myogenic markers for non-mammalian cells can be different than those described here.  
1.3 This guide proposes appropriate markers to measure when conducting myogenic differentiation assays. This guide describes the stages for multipotent stem cell differentiation toward myoblasts and myotubes. This guide provides information about the appropriate methods to determine myogenic differentiation. This guide does not provide information about media, supplements, or substrates that drive differentiation toward a myogenic phenotype.  
1.4 The purpose of this guide is to act as an aid for work performed in the area of skeletal myogenesis. Using this guide, researchers should be able to understand which skeletal muscle markers are best suited for experiments. This guide will improve consistency for studies of myogenic differentiation of multipotent stem cells by identifying appropriate markers for each stage leading to myocyte differentiation. It should be noted that myoblast differentiation in vitro may not be predictive of results that may be obtained in vivo.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Gu...

SIGNIFICANCE AND USE
4.1 The main use is to immobilize, support, or suspend living cells or tissue in a matrix. The use of an encapsulation/immobilization system may protect cells or tissues from immune rejection. When immobilizing biological material in alginate gels, there are numerous parameters that must be controlled. This guide contains a list of these parameters and describes the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of an encapsulation system using alginate. This guide only covers single gelled beads, coated or not, and not double capsules or other constructs.  
4.2 The alginate gelation technology covered by this guide may allow the formulation of cells and tissues into biomedical devices for use as tissue engineered medical products or drug delivery devices. These products may be appropriate for implantation based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application.
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1.1 This guide discusses information relevant to the immobilization or encapsulation of living cells or tissue in alginate gels. Immobilized or encapsulated cells are suitable for use in biomedical and pharmaceutical applications, or both, including, but not limited to, Tissue Engineered Medical Products (TEMPs).  
1.2 This guide addresses key parameters relevant for successful immobilization and encapsulation in alginate gels.  
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.5 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 This guide is designed to assist medical device manufacturers as they develop new devices or qualify existing devices (e.g., catheters, needles) for delivering clinical cell therapies. Cytocompatibility considers the impact of the delivery device on the cells passing through the device during the delivery procedure. The biological safety of the device (e.g., the device’s cytotoxicity) should be addressed via other methods, such as ISO 10993-5. It is understood that this guide does not address testing of specific cellular products with specific delivery devices. Such testing may be required by regulatory authorities prior to clinical trial of cellular product or marketing applications. This guide outlines considerations to make the product qualification procedures more likely to succeed and more cost effective.  
4.2 The key aspects of assessing device cytocompatibility include selecting a test cell line or cell lines and determining the cell physiology parameters that will be measured to make a determination of cytocompatibility. Acceptance criteria for designating a device as cytocompatible are not detailed here. It will be up to the delivery device end user to determine if the results of a cytocompatibility assessment are sufficient to consider that device cytocompatible. Delivery device lot to lot variability may impact cytocompatibility, therefore validated manufacturing processes should be considered when producing devices for cytocompatibility assessments.
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1.1 This guide outlines the parameters to consider when designing in vitro tests to assess the potential impact of a delivery device on a cellular product being dispensed. This guide does not provide specific protocols, but rather suggests what should be considered the minimum characterization necessary to assess device cytocompatibility. Topics discussed include selecting an appropriate cell line(s), cell physiology parameters to measure, and relevant test procedure variables. Only cells suspended in liquid and infused through a device are considered. Cell therapies paired with scaffolds, suspended in hydrogels, or administered via other methods (e.g., tissue grafting) are not included in the scope of this document. This document does not address physical characterization of delivery devices, such as mechanics, composition, or degradation.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
4.1 Autologous PRP and platelet gels are utilized in a wide range of orthopedic, sports medicine, regenerative medicine, and surgical applications (3-5). PRP and platelet gels are layered, sprayed, injected, molded, or packed, alone or in combination with graft material or TEMPs, into a variety of anatomical sites, tissues, and voids (3, 6). These platelet concentrates can provide an assortment of bioactive molecules, cells, and physical properties that are potentially attractive for promoting healing and other cell therapy applications (7). Unfortunately, the term “platelet-rich plasma” or “PRP,” which is ubiquitous in early and contemporary medical literature related to a variety of platelet concentrates, only unambiguously denotes one critical parameter of a platelet suspension—increased platelet concentration. Without further context, this common description of PRP offers no information about other important physical and cellular aspects of platelet concentrations. As scientific and clinical understanding of PRP and other cellular therapies increases standardization of nomenclature and terminology is critical for defining key properties, standardizing processing parameters and techniques, and developing repeatable assays for quality assurance and scientific evaluation (5, 8-13). This guide outlines basic guidelines to describe key properties of unique PRP and platelet gel formulations in a standardized fashion. Reliable, standardized descriptions can provide valuable context to PRP end users, such as clinicians seeking a PRP or platelet gel with certain biological attributes or scientific investigators seeking to duplicate a published formulation or to correlate a given PRP or platelet gel feature to other biological properties or outcomes.
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1.1 This guide defines terminology and identifies key fundamental properties of autologous platelet-rich plasma (PRP) and PRP-derived platelet gels intended to be used for tissue engineered medical products (TEMPS) or for cell therapy applications. This guide provides a common nomenclature and basis for describing notable properties and processing parameters for PRP and platelet gels that may have utility for manufacturers, researchers, and clinicians. Further discussion is also provided on certain aspects of PRP processing techniques, characterization, and quality assurance and how those considerations may impact key properties. The PRP characteristics outlined in this guide were selected based n a review of contemporary scientific and clinical literature but do not necessarily represent a comprehensive inventory; other significant unidentified properties may exist or be revealed by future scientific evaluation. This guide provides general recommendations for how to identify and cite relevant characteristics of PRP, based on broad utility; however, users of this standard should consult referenced documents for further information on the relative import or significance of any particular PRP characteristic in a particular context.  
1.2 The scope of this guide is confined to aspects of PRP and platelet gels derived and processed from autologous human peripheral blood. Platelet-rich plasma, as defined within the scope of this standard, may include leukocytes.  
1.3 The scope of this document is limited to guidance for PRP and platelet gels that are intended to be used for TEMPS or for cell therapy applications. Processing of PRP, other platelet concentrates or other blood components for direct intravenous transfusion is outside the scope of this guide. Apheresis platelets and other platelet concentrates utilized in transfusion medicine are outside the scope of this document. Production of PRP or platelet gels for diagnostic or research applications unrelated to PRP intended for TEMPS or cell therapy is also outside the scope of this guide. Fibrin gels devoid of platelets are also excluded from discussion within this document.  
1.4 This standar...

SIGNIFICANCE AND USE
3.1 The electrical sensing zone method for cell counting is used in tissue culture, government research, and hospital, biomedical, and pharmaceutical laboratories for counting and sizing cells. The method may be applicable to a wide range of cells sizes and cell types, with appropriate validation (10).  
3.2 The electrical sensing zone methodology was introduced in the mid-1950s (9). Since this time, there have been substantial improvements which have enhanced the operator's ease of use. Among these are the elimination of the mercury manometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.  
3.3 This instrumentation offers a rapid result as contrasted to the manual counting of cells using the hemocytometer standard counting chamber. The counting chamber is known to have an error of 10 to 30 %, as well as being time-consuming (11). In addition, when counting and sizing porcine hepatocytes, Stegemann et al concluded that the automated, electrical sensing zone method provided greater accuracy, precision, and speed, for both counts and size, compared to the conventional microscopic or the cell mass-based method (7).
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1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings and is applicable to a wide range of cell types. The method works best for spherical cells, and may be less accurate if cells are not spherical, such as for discoid cells or budding yeast. The method is appropriate for suspension as well as adherent cell cultures (1).2 Results may be reported as number of cells per milliliter or total number of cells per volume of cell suspension analyzed. Size distribution may be expressed in cell diameter or volume.  
1.2 Cells commonly used in tissue-engineered medical products (2) are analyzed routinely. Examples are chondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al. used the method for both pancreatic islet number and volume measurements (6). In addition, instrumentation using the electrical sensing zone technology was used for both count and size distribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (7), and others (6, 8), the automated electrical sensing zone method was validated for precision when compared to the conventional visual cell counting under a microscope using a hemocytometer. Currently, it is not possible to validate cell counting devices for accuracy, since there not a way to produce a reference sample that has a known number of cells. The electrical sensing zone method shall be validated each time it is implemented in a new laboratory, it is used on a new cell type, or the cell counting procedure is modified.  
1.3 Electrical sensing zone instrumentation (commonly referred to as a Coulter counter) is manufactured by a variety of companies and is based upon electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see Fig. 1(9)). When cells are suspended in a conductive liquid, phosphate-buffered saline for instance, they function as discrete insulators. When the cell suspension is drawn through a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submerged electrodes located on each side of the aperture. An electrical pulse, suitable for both counting and sizing, results from the passage of each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the “electronic sensing zone.” This test method permits the selective counting of cells within narrow size distrib...

SIGNIFICANCE AND USE
4.1 Cell attachment or, lack of it, to biomaterials is a critical factor affecting the performance of a device or implant. Cell attachment is a complicated, time-dependent, process involving significant morphological changes of the cell and deposition of a bed of extracellular matrix. Details of the adhesive bond that is formed have been reviewed by, for example, Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and Garcia and Gallant (2003) (6). The strength of this coupling can be determined either by monitoring the force of attachment between a cell and a substrate over time or by measuring the force required to detach the cell once it has adhered.  
4.2 Cell adhesion to a surface depends on a range of biological and physical factors that include the culture history, the age of the cell, the cell type, and both the chemistry and morphology of the underlying surface and time. These elements need to be considered in developing a test protocol.  
4.3 Devising robust methods for measuring the propensity of cells to attach to different substrates is further complicated since either cell adhesion or detachment can be assessed. These processes are not always similar or complementary.  
4.4 Most studies of cell attachment focus on obtaining some measure of the time-dependent force required to detach, or de-adhere, cells that have already adhered to a surface (James et al, 2005) (7). More recently investigators have begun to measure the adhesive forces that develop between cells and the underlying surface during attachment (Lukas and Dvorak, 2004) (5). From a practical point of view, it is much easier to measure the force required to detach or de-adhere cells from a surface than to measure those that develop during attachment. However, in both cases, the experimental data should be interpreted with a degree of caution that depends on the intended use of the measurements. The methods of measuring cell adhesion described herein are measures of the force required to ...
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1.1 This guide describes protocols that can be used to measure the strength of the adhesive bond that develops between a cell and a surface as well as the force required to detach cells that have adhered to a substrate. Controlling the interactions of mammalian cells with surfaces is fundamental to the development of safe and effective medical products. This guide does not cover methods for characterizing surfaces. The information generated by these methods can be used to obtain quantitative measures of the susceptibility of surfaces to cell attachment as well as measures of the adhesion of cells to a surface. This guide also highlights the importance of cell culture history and influences of cell type.  
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.3 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

SIGNIFICANCE AND USE
5.1 This guide describes markers involved in myoblast differentiation that can be used to screen stem cells to help define myogenic capacity. Stem cells include pluripotent and multipotent stem cells capable of differentiating into several different mesenchymal cells, including skeletal muscle myoblasts.  
5.2 To assess myogenesis in cells derived and not derived from muscle, markers are measured to accurately define the changes in transcription and structural proteins that regulate differentiation, fusion, and myotube formation. Discussion of these markers is important to understand why they are recommended.  
5.3 Myogenic Differentiation:  
5.3.1 Myogenic differentiation is a highly regulated process controlled by paired box (Pax) transcription factors and the myogenic regulatory factor (MRF) family. During early differentiation in adults, myogenic progenitors such as activated satellite cells or myoblasts express Pax3 and Pax7. Pax3 and Pax7 transcription factors switch the cells toward a myogenic fate, and repress myocyte differentiation (2), priming the cell for later MRFs. To form muscle, the family of MRFs is required to terminally differentiate myoblasts and form myofibers. These regulatory proteins belong to a superfamily of basic helix-loop-helix transcription factors that consists of myogenic differentiation factor 1 (Myod1), myogenic factor 5 (Myf5), myogenin (Myog), and myogenic factor 6 (Myf6). In the initial stages of myogenic differentiation, Myod1 and Myf5 are the first MRFs to be expressed, and trigger increased production of Myog and Myf6  (3). Increased intracellular Myog and Myf6 induces terminal differentiation of myoblasts into myocytes, leading to fused myotubes.  
5.4 Forming Myotubes:  
5.4.1 While myogenic markers describe differentiation, fusion into multinucleated myotubes is an important factor in muscle biology. Myoblasts differentiate into a fusogenic phenotype characterized by multiple fusion markers. One marker of note is m-c...
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1.1 Myogenic differentiation is a process regulated by specific transcription factors and signaling molecules that have been shown to induce a myogenic phenotype. Transcription factors mark the stages of myogenesis and act as benchmarks for use in myogenic assays.  
1.2 This guide applies to mammalian cells but does not apply to non-mammalian cells as the myogenic markers for non-mammalian cells can be different than those described here.  
1.3 This guide proposes appropriate markers to measure when conducting myogenic differentiation assays. This guide describes the stages for multipotent stem cell differentiation toward myoblasts and myotubes. This guide provides information about the appropriate methods to determine myogenic differentiation. This guide does not provide information about media, supplements, or substrates that drive differentiation toward a myogenic phenotype.  
1.4 The purpose of this guide is to act as an aid for work performed in the area of skeletal myogenesis. Using this guide, researchers should be able to understand which skeletal muscle markers are best suited for experiments. This guide will improve consistency for studies of myogenic differentiation of multipotent stem cells by identifying appropriate markers for each stage leading to myocyte differentiation. It should be noted that myoblast differentiation in vitro may not be predictive of results that may be obtained in vivo.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Gu...

SIGNIFICANCE AND USE
5.1 The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine in vivo product performance of cell/biomaterial constructs (see 5.7); therefore there is a need for standardized test methods to quantify cell viability.  
5.2 There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells onto scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell numbers within a given methodology.  
5.3 As described in Guide F2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantified using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.  
5.4 These test methods can be used to quantify cells on hard or soft 3-D biomaterials, such as ceramics and polymer gels. The test methods also apply to cells seeded on porous coatings.  
5.5 Test methods described in this guide may also be used to distinguish between proliferating and non-proliferating viable cells. Proliferating cells proceed through the DNA synthesis (S) pha...
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1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue-engineered medical products (TEMPs).  
1.2 In addition to providing a compendium of available techniques, this guide describes materials-specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid, and/or account for, scaffold material/cell viability assay interactions.  
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.  
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.  
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This guidance document describes the components and conditions used for in vitro osteoblast differentiation assays that can be used to screen for the osteogenic capability of progenitor stem cells from various human or animal sources, including mixed tissue-derived connective tissue progenitor populations, or cell populations that may be selectively isolated or manipulated through culture expansion, processing, transfection or genetic modification.  
4.2 The osteoblast differentiation assay may be referred to as an osteogenesis assay or a mineralization assay.  
4.3 It is important to carefully select the components and conditions used for in vitro osteoblast differentiation assays since high amounts of osteogenic medium components can lead to dystrophic, pathologic or artifactual calcium-based precipitates that do not indicate differentiation of the cells in culture to functional osteoblasts (1).3 For example, when high concentrations of beta-glycerophosphate are used in the medium to function as a substrate for the enzyme alkaline phosphatase secreted by the cells, there is a marked increase in free phosphate, which then precipitates with Ca++ ions in the media to form calcium phosphate crystals independently of the differentiation status of the progenitor cell (2, 3).  
4.4 Alkaline phosphatase production is an early event associated with osteoblast differentiation but it can also be stimulated in other cell types by the addition of the osteogenic supplement dexamethasone to the medium. Alkaline phosphatase enhances the formation of calcified deposits prior to their natural occurrence in bone that typically coincides with bone sialoprotein and osteocalcin expression by mineralized matrix-producing osteoblasts. These kinds of calcified/mineral deposits are thus considered dystrophic, pathologic, or artifactual because they were not initiated by a mature osteoblast. A calcium measurement, such as that described in Practice F2997 for the Quantification of Cal...
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1.1 This document provides guidance on how to conduct in vitro osteoblast differentiation assays with progenitor stem cells including mesenchymal stromal cells.  
1.2 This document describes the roles of various osteogenic supplements that are added to the cell culture medium of an osteoblast differentiation assay to encourage and support the differentiation of progenitor cells into matrix-producing osteoblasts.  
1.3 This document provides recommendations for the concentrations of osteogenic supplements that may prevent the precipitation of artifactual mineral deposits that are not directly produced by osteoblasts, nor correlated with osteoblastic gene expression of the cells.  
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
4.1 This test method describes a cell adhesion method that can be used to provide a detachment percent at a given RCF for cells that have adhered to a substrate, typically for a short time. The information generated by this test method can be used to obtain a semi-quantitative measurement of the adhesion of cells to either an uncoated or pre-coated substrate, when compared to a reference (adherent) cell type on the same substrate. As described in Reyes and Garcia (2003), it is recommended that the 50 % point be used for either ligand concentration or RCF for the most robust measurement of adhesion strength. The adhesion may vary due to changes in the phenotype of the cells or as a result of the specific properties of the surface. The substrate may include tissue culture-treated polystyrene, biomaterials or bioactive surfaces. If the substrate is a hydrogel, care must be taken to avoid cohesive failure in the hydrogel (that is, detached cells have pulled away fragments of gel). The coating may consist of (but is not limited to) the following: natural or synthetic biomaterials, hydrogels, components of extracellular matrix (ECM), ligands, adhesion or bioactive molecules, genes or gene products. Cell concentration is also critical, as use of too high a concentration of cells may result in cells detaching as a sheet, rather than as individual cells. This centrifugation approach, once validated, may be applicable for quality control (QC) and product development. However, until the method is correlated to other measures of cell attachment, the current method should be run in parallel with other known measures of cell adhesion.  
4.2 This test method does not cover test methods to quantitate changes in gene expression, or changes in biomarkers, as identified by immunostaining. This test method additionally does not cover quantitative image analysis techniques. In some cases the change in adhesive properties may reflect on the degree of differentiation or de-differentiatio...
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1.1 This test method covers a centrifugation cell adhesion assay that can be used to detect changes in adhesive characteristics of cells with passage or treatments. This approach measures the force required to detach cells from a substrate. Adhesion, among many variables, may vary due to changes in the phenotype of the cells.  
1.2 This test method does not cover methods to verify the uniformity of coating of surfaces, nor does it cover methods for characterizing surfaces.  
1.3 The cells may include adult, progenitor or stem cells from any species. The types of cells may include chondrocytes, fibroblasts, osteoblast, islet cells, or other relevant adherent cell types.  
1.4 This test method does not cover methods for isolating or harvesting of cells. This test method does not cover test methods to quantitate changes in gene expression, or changes in biomarker type or concentration, as identified by immunostaining. Nor does this test method cover quantitative image analysis techniques.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
5.1 In-vitro osteoblast differentiation assays are one approach to screen progenitor stem cells for their capability to become osteoblasts. The extent of calcified deposits or mineralized matrix that form in-vitro may be an indicator of differentiation to a functional osteoblast; however, gene expression of osteogenic genes or proteins is another important measurement to use in conjunction with this assay to determine the presence of an osteoblast.  
5.2 This test method provides a technique for staining, imaging, and quantifying the fluorescence intensity and area related to the mineralization in living cell cultures using the non-toxic calcium-chelating dye, xylenol orange. The positively stained area of mineralized deposits in cell cultures is an indirect measure of calcium content. It is important to measure the intensity to assure that the images have not been underexposed or overexposed. Intensity does not correlate directly to calcium content as well as area.  
5.3 Xylenol orange enables the monitoring of calcified deposits repeatedly throughout the life of the culture without detriment to the culture. There is no interference on subsequent measurements of mineralized area due to dye accumulation from repeated application (1).3 Calcified deposits that have been previously stained may appear brighter, but this does not impact the area measurement. Calcein dyes may also be used for this purpose (1) but require a different procedure for analysis than xylenol orange (i.e., concentration and filter sets) and are thus not included here. Alizarin Red and Von Kossa are not suitable for use with this procedure on living cultures since there is no documentation supporting their repeated use in living cultures without deleterious effects.  
5.4 The test method may be applied to cultures of any cells capable of producing calcified deposits. It may also be used to document the absence of mineral in cultures where the goal is to avoid mineralization.  
5.5 During osteo...
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1.1 This practice defines a method for the estimation of calcium content at multiple time points in living cell cultures that have been cultured under conditions known to promote mineralization. The practice involves applying a fluorescent calcium chelating dye that binds to the calcium phosphate mineral crystals present in the live cultures followed by image analysis of fluorescence microscopy images of the stained cell cultures. Quantification of the positively stained areas provides a relative measure of the calcium content in the cell culture plate. A precise correlation between the image analysis parameters and calcium content is beyond the scope of this practice.  
1.2 Calcium deposition in a secreted matrix is one of several features that characterize bone formation (in vitro and in vivo), and is therefore a parameter that may indicate bone formation and osteoblast function (i.e., osteoblastic differentiation). Calcium deposition may, however, be unrelated to osteoblast differentiation status if extensive cell death occurs in the cell cultures or if high amounts of osteogenic medium components that lead to artifactual calcium-based precipitates are used. Distinguishing between calcium deposition associated with osteoblast-produced mineralized matrix and that from pathological or artifactual deposition requires additional structural and chemical characterization of the mineralized matrix and biological characterization of the cell that is beyond the scope of this practice.  
1.3 The parameters obtained by image analysis are expressed in relative fluorescence units or area percentage, e.g., fraction of coverage of the area analyzed.  
1.4 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of ...

SIGNIFICANCE AND USE
The Manual Observer-Dependent Assay—The manual quantification of cell and CFU cultures based on observer-dependent criteria or judgment is an extremely tedious and time-consuming task and is significantly impacted by user bias. In order to maintain consistency in data acquisition, pharmacological and drug discovery and development studies utilizing cell- and colony-based assays often require that a single observer count cells and colonies in hundreds, and potentially thousands of cultures. Due to observer fatigue, both accuracy and reproducibility of quantification suffer severely (5). When multiple observers are employed, observer fatigue is reduced, but the accuracy and reproducibility of cell and colony enumeration is still significantly compromised due to observer bias and significant intra- and inter-observer variability (4, 13). Use of quantitative automated image analysis provides data for both the number of colonies as well as the number of cells in each colony. These data can also be used to calculate mean cells per colony. Traditional methods for quantification of colonies by hand counting coupled with an assay for cell number (for example, DNA or mitochondrial) remains a viable method that can be used to calculate the mean number of cells per colony. These traditional methods have the advantage that they are currently less labor intensive and less technically demanding (8, 9). However, the traditional assays do not, provide colony level information (for example, variation and skew), nor do they provide a means for excluding cells that are not part of a colony from the calculation of mean colony size. As a result, the measurement of the mean number of cells per colony that is obtained from these alternative methods may differ when substantial numbers of cells in a sample are not associated with colony formation. By employing state-of-the-art image acquisition, processing and analysis hardware and software, an accurate, precise, robust and automated analysi...
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1.1 This test method, provided its limitations are understood, describes a procedure for quantitative measurement of the number and biological characteristics of colonies derived from a stem cell or progenitor population using image analysis.
1.2 This test method is applied in an in vitro laboratory setting.
1.3 This method utilizes: (a) standardized protocols for image capture of cells and colonies derived from in vitro processing of a defined population of starting cells in a defined field of view (FOV), and (b) standardized protocols for image processing and analysis.
1.4 The relevant FOV may be two-dimensional or three-dimensional, depending on the CFU assay system being interrogated.
1.5 The primary unit to be used in the outcome of analysis is the number of colonies present in the FOV. In addition, the characteristics and sub-classification of individual colonies and cells within the FOV may also be evaluated, based on extant morphological features, distributional properties, or properties elicited using secondary markers (for example, staining or labeling methods).
1.6 Imaging methods require that images of the relevant FOV be captured at sufficient resolution to enable detection and characterization of individual cells and over a FOV that is sufficient to detect, discriminate between, and characterize colonies as complete objects for assessment.
1.7 Image processing procedures applicable to two- and three-dimensional data sets are used to identify cells or colonies as discreet objects within the FOV. Imaging methods may be optimized for multiple cell types and cell features using analytical tools for segmentation and clustering to define groups of cells related to each other by proximity or morphology in a manner that is indicative of a shared lineage relationship (that is, clonal expansion of a single founding stem cell or progenitor).
1.8 The characteristics of individual colony objects (cells per...

SIGNIFICANCE AND USE
Cell attachment or, lack of it, to biomaterials is a critical factor affecting the performance of a device or implant. Cell attachment is a complicated, time-dependent, process involving significant morphological changes of the cell and deposition of a bed of extracellular matrix. Details of the adhesive bond that is formed have been reviewed by, for example, Pierres et al (2002) (4), Lukas and Dvorak (2004) (5), and Garcia and Gallant (2003) (6). The strength of this coupling can be determined either by monitoring the force of attachment between a cell and a substrate over time or by measuring the force required to detach the cell once it has adhered.
Cell adhesion to a surface depends on a range of biological and physical factors that include the culture history, the age of the cell, the cell type, and both the chemistry and morphology of the underlying surface and time. These elements that need to be considered in developing a test protocol.
Devising robust methods for measuring the propensity of cells to attach to different substrates is further complicated since either cell adhesion or detachment can be assessed. These processes that are not always similar or complementary.
Most studies of cell attachment focus on obtaining some measure of the time-dependent force required to detach, or de-adhere, cells that have already adhered to a surface (James et al, 2005) (7). More recently investigators have begun to measure the adhesive forces that develop between cells and the underlying surface during attachment (Lukas and Dvorak, 2004) (5). From a practical point of view, it is much easier to measure the force required to detach or de-adhere cells from a surface than to measure those that develop during attachment. However, in both cases, the experimental data should be interpreted with a degree of caution that depends on the intended use of the measurements. The methods of measuring cell adhesion described herein are measures of the force required to detach an...
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1.1 This guide describes protocols that can be used to measure the strength of the adhesive bond that develops between a cell and a surface as well as the force required to detach cells that have adhered to a substrate. Controlling the interactions of mammalian cells with surfaces is fundamental to the development of safe and effective medical products. This guide does not cover methods for characterizing surfaces. The information generated by these methods can be used to obtain quantitative measures of the susceptibility of surfaces to cell attachment as well as measures of the adhesion of cells to a surface. This guide also highlights the importance of cell culture history and influences of cell type.
1.2 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
The main use is to immobilize, support, or suspend living cells or tissue in a matrix. The use of an encapsulation/immobilization system may protect cells or tissues from immune rejection. When immobilizing biological material in alginate gels, there are numerous parameters that must be controlled. This guide contains a list of these parameters and describes the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of an encapsulation system using alginate. This guide only covers single gelled beads, coated or not, and not double capsules or other constructs.
The alginate gelation technology covered by this guide may allow the formulation of cells and tissues into biomedical devices for use as tissue engineered medical products or drug delivery devices. These products may be appropriate for implantation based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application.
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1.1 This guide discusses information relevant to the immobilization or encapsulation of living cells or tissue in alginate gels. Immobilized or encapsulated cells are suitable for use in biomedical and pharmaceutical applications, or both, including, but not limited to, Tissue Engineered Medical Products (TEMPs).
1.2 This guide addresses key parameters relevant for successful immobilization and encapsulation in alginate gels.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

SIGNIFICANCE AND USE
The main use is to immobilize, support, or suspend living cells or tissue in a matrix. The use of an encapsulation/immobilization system may protect cells or tissues from immune rejection. When immobilizing biological material in alginate gels, there are numerous parameters that must be controlled. This guide contains a list of these parameters and describes the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of an encapsulation system using alginate. This guide only covers single gelled beads, coated or not, and not double capsules or other constructs.
The alginate gelation technology covered by this guide may allow the formulation of cells and tissues into biomedical devices for use as tissue engineered medical products or drug delivery devices. These products may be appropriate for implantation based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application.
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1.1 This guide discusses information relevant to the immobilization or encapsulation of living cells or tissue in alginate gels. Immobilized or encapsulated cells are suitable for use in biomedical and pharmaceutical applications, or both, including, but not limited to, Tissue Engineered Medical Products (TEMPs).
1.2 This guide addresses key parameters relevant for successful immobilization and encapsulation in alginate gels.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

SIGNIFICANCE AND USE
The number and distribution of viable and non-viable cells within, or on the surface of, a biomaterial scaffold is one of several important characteristics that may determine in vivo product performance of cell/biomaterial constructs (see 5.7); therefore there is a need for standardized test methods to quantitate cell viability.
There are a variety of static and dynamic methods to seed cells on scaffolds, each with different cell seeding efficiencies. In general, static methods such as direct pipetting of cells on to scaffold surfaces have been shown to have lower cell seeding efficiencies than dynamic methods that push cells into the scaffold interior. Dynamic methods include: injection of cells into the scaffold, cell seeding on biomaterials contained in spinner flasks or perfusion chambers, or seeding that is enhanced by the application of centrifugal forces. The methods described in this guide can assist in establishing cell seeding efficiencies as a function of seeding method and for standardizing viable cell number within a given methodology.
As described in Guide F 2315, thick scaffolds or scaffolds highly loaded with cells lead to diffusion limitations during culture or implantation that can result in cell death in the center of the construct, leaving only an outer rim of viable cells. Spatial variations of viable cells such as this may be quantitated using the tests within this guide. The effectiveness of the culturing method or bioreactor conditions on the viability of the cells throughout the scaffold can also be evaluated with the methods described in this guide.
These test methods can be used to quantitate cells on hard or soft 3-D biomaterials, such as ceramics and polymer gels. The test methods also apply to cells seeded on porous coatings.
Test methods described in this guide may also be used to distinguish between proliferating and non-proliferating viable cells. Proliferating cells proceed through the DNA synthesis (S) phase and the mitosis...
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1.1 This guide is a resource of cell viability test methods that can be used to assess the number and distribution of viable and non-viable cells within porous and non-porous, hard or soft biomaterial scaffolds, such as those used in tissue engineered medical products (TEMPs).
1.2 In addition to providing a compendium of available techniques, this guide describes materials specific interactions with the cell assays that can interfere with accurate cell viability analysis, and includes guidance on how to avoid, and/or account for, scaffold material/cell viability assay interactions.
1.3 These methods can be used for 3-D scaffolds containing cells that have been cultured in vitro or for scaffold/cell constructs that are retrieved after implantation in living organisms.
1.4 This guide does not propose acceptance criteria based on the application of cell viability test methods.
1.5 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
This assay is used in university tissue culture laboratories, government research, and hospital, biomedical, and pharmaceutical laboratories to automate cell counting and sizing. This instrumentation provides very rapid, accurate, and precise results for any tissue culture facility. In addition, as noted, since the cell sizes to be analyzed by the instrument are set by the user, the analyses may be done on virtually any species of cells and cell type; it is not restricted to human cells or blood cells.
The electrical sensing zone methodology was introduced in the mid 1950s (9). Since this time, there have been substantial improvements which have enhanced the operator’ease of use. Among these are the elimination of the mercury manometer, reduced size, greater automation, and availability of comprehensive statistical computer programs.
This instrumentation offers a rapid result as contrasted to the manual counting of cells using the standard counting chamber, hemocytometer. The counting chamber is known to have an error of 10 to 30 %, as well as being very time consuming (10). In addition, when counting and sizing porcine hepatocytes, Stegemann et al concluded that the automated, electrical sensing zone method provided significantly greater accuracy, precision, and speed, for both counts and size, compared to the conventional microscopic or the cell mass-based method (7).
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1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings, hence, this test method is not restricted to specific cell types. The method is appropriate for suspension as well as adherent cell cultures (). This is a quantitative laboratory method not intended for on-line or field use. Results may be reported as number of cells per millilitre or total number of cells per volume of cell suspension analyzed. Both count and size distribution may be expressed in cell micron diameter or volume, femtolitres.
1.2 Cells commonly used in tissue-engineered medical products () routinely are analyzed. Examples are chondrocytes (), fibroblasts (), and keratinocytes (). Szabo et al used the method for both pancreatic islet number and volume measurements (). In addition, instrumentation using the electrical sensing zone technology was used for both count and size distribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (), and others (, ), the automated electrical sensing zone method was clearly validated for superior accuracy and precision when compared to the conventional manual method, visual cell counting under a microscope using a hemocytometer. This validation has been demonstrated over a wide variety of cell types. In addition, the automated procedure is rapid, rugged, and cost effective; it also minimizes operator-to-operator variability inherent in manual techniques.
1.3 This instrumentation is manufactured by a variety of companies; however, the principle used in all is electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see ()). When cells are suspended in a conductive liquid, phosphate-buffered saline for instance, they function as discrete insulators. When the cell suspension is drawn through a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submerged electrodes located on each side of the aperture. An electrical pulse, suitable for both counting and sizing, results from the passage of each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the "electronic sensing zone." This test...

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1.1 This guide describes protocols that can be used to measure the strength of the adhesive bond that develops between a cell and a surface as well as the force required to detach cells that have adhered to a substrate. Controlling the interactions of mammalian cells with surfaces is fundamental to the development of safe and effective medical products. This guide does not cover methods for characterizing surfaces. The information generated by these methods can be used to obtain quantitative measures of the susceptibility of surfaces to cell attachment as well as measures of the adhesion of cells to a surface. This guide also highlights the importance of cell culture history and influences of cell type.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

SIGNIFICANCE AND USE
The main use is to immobilize, support, or suspend living cells or tissue in a matrix. The use of an encapsulation/immobilization system may protect cells or tissues from immune rejection. When immobilizing biological material in alginate gels, there are numerous parameters that must be controlled. This guide contains a list of these parameters and describes the methods and types of testing necessary to properly characterize, assess, and ensure consistency in the performance of an encapsulation system using alginate. This guide only covers single gelled beads, coated or not, and not double capsules or other constructs.
The alginate gelation technology covered by this guide may allow the formulation of cells and tissues into biomedical devices for use as tissue engineered medical products or drug delivery devices. These products may be appropriate for implantation based on supporting biocompatibility and physical test data. Recommendations in this guide should not be interpreted as a guarantee of clinical success in any tissue engineered medical product or drug delivery application.
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DESIG: F2315 03 ^TITLE: Standard Guide for Immobilization or Encapsulation of Living Cells or Tissue in Alginate Gels ^SCOPE:1. Scope
1.1 This guide discusses information relevant to the immobilization or encapsulation of living cells or tissue in alginate gels. Immobilized or encapsulated cells are suitable for use in biomedical and pharmaceutical applications, or both, including, but not limited to, Tissue Engineered Medical Products (TEMPs).
1.2 This guide addresses key parameters relevant for successful immobilization and encapsulation in alginate gels.
This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

SIGNIFICANCE AND USE
This guide describes the general product development criteria and analyses applicable to processing of cells, tissues, and organs used for the production of TEMPs. For the purposes of this guide, cells, tissues, and organs may be derived from any organism at any developmental stage and in any state of health. For example, this guide applies to stem, progenitor, somatic, and germline cells, as well as cells from specific tissue and organ types. This guide also applies to cells, tissues, and organs from healthy, diseased, or injured embryos to adults.
Cells, tissues, and organs may be combined with a scaffold and may contain locally or systemically acting biomolecules or a drug (medicinal) product. This type of TEMP would be a “combination product.”
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1.1 This guide describes the processing, characterization, production, and quality assurance of cells, tissues, and organs used for Tissue Engineered Medical Products (TEMPs). It concerns aspects of processing activities for cells, tissues, and organs to be further processed. These aspects include: (1) cell, tissue, and organ processing (that is, facility, reagents, and procedures for receipt, inspection, and storage; tissue culture components, biological risk factors, and processing area), (2) donors (human and nonhuman) and screening, and (3) cell, tissue, and organ characterization and processing.
1.2 This guide does not apply to any medical products of human origin regulated by the U.S. Food and Drug Administration (FDA) under 21 CFR Parts 16 and 1270 (1) and 21 CFR Parts 207, 807, and 1271 (2).
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.

SCOPE
1.1 This test method, provided the limitations are understood, covers a procedure for both the enumeration and measurement of size distribution of most all cell types. The instrumentation allows for user-selectable cell size settings, hence, this test method is not restricted to specific cell types. The method is appropriate for suspension as well as adherent cell cultures (1). This is a quantitative laboratory method not intended for on-line or field use. Results may be reported as number of cells per millilitre or total number of cells per volume of cell suspension analyzed. Both count and size distribution may be expressed in cell micron diameter or volume, femtolitres.
1.2 Cells commonly used in tissue-engineered medical products (2) routinely are analyzed. Examples are chondrocytes (3), fibroblasts (4), and keratinocytes (5). Szabo et al used the method for both pancreatic islet number and volume measurements (6). In addition, instrumentation using the electrical sensing zone technology was used for both count and size distribution analyses of porcine hepatocytes placed into hollow fiber cartridge extracorporeal liver assist systems. In this study (7), and others (6, 8), the automated electrical sensing zone method was clearly validated for superior accuracy and precision when compared to the conventional manual method, visual cell counting under a microscope using a hemocytometer. This validation has been demonstrated over a wide variety of cell types. In addition, the automated procedure is rapid, rugged, and cost effective; it also minimizes operator-to-operator variability inherent in manual techniques.
1.3 This instrumentation is manufactured by a variety of companies; however, the principle used in all is electrical impedance. This test method, for cell counting and sizing, is based on the detection and measurement of changes in electrical resistance produced by a cell, suspended in a conductive liquid, traversing through a small aperture (see (Fig. 1)). When cells are suspended in a conductive liquid, phosphate-buffered saline for instance, they function as discrete insulators. When the cell suspension is drawn through a small cylindrical aperture, the passage of each cell changes the impedance of the electrical path between two submerged electrodes located on each side of the aperture. An electrical pulse, suitable for both counting and sizing, results from the passage of each cell through the aperture. The path through the aperture, in which the cell is detected, is known as the "electronic sensing zone." This test method permits the selective counting of cells within very narrow size distribution ranges by electronic selection of the generated pulses. While the number of pulses indicates cell count, the amplitude of the electrical pulse produced depends on the cell's volume. The baseline resistance between the electrodes is due to the resistance of the conductive liquid within the boundaries of the aperture. The presence of cells within the "electronic sensing zone" raises the resistance of the conductive pathway that depends on the volume of the cell. Analyses of the behavior of cells within the aperture demonstrates that the height of the pulse produced by the cell is the parameter that most nearly shows proportionality to the cell volume.
1.4 Limitations are discussed as follows:
1.4.1 Coincidence-Occasionally, more than a single cell transverses the aperture simultaneously. Only a single larger pulse, as opposed to two individual pulses, is generated. The result is a lower cell count and higher cell volume measurement. The frequency of coincidence is a statistically predictable function of cell concentration that is corrected by the instrument. This is called coincidence correction (8). This phenomenon may be minimized, thus ensuring greater result accuracy, by using relatively low cell concentrations, around the 5 % level.
1.4.2 Viability-Automated cell counting enumerates both viable ...

SIGNIFICANCE AND USE
This guide describes the general product development criteria and analyses applicable to processing of cells, tissues, and organs used for the production of TEMPs. For the purposes of this guide, cells, tissues, and organs may be derived from any organism at any developmental stage and in any state of health. For example, this guide applies to stem, progenitor, somatic, and germline cells, as well as cells from specific tissue and organ types. This guide also applies to cells, tissues, and organs from healthy, diseased, or injured embryos to adults.
Cells, tissues, and organs may be combined with a scaffold and may contain locally or systemically acting biomolecules or a drug (medicinal) product. This type of TEMP would be a “combination product.”
SCOPE
1.1 This guide describes the processing, characterization, production, and quality assurance of cells, tissues, and organs used for Tissue Engineered Medical Products (TEMPs). It concerns aspects of processing activities for cells, tissues, and organs to be further processed. These aspects include: (1) cell, tissue, and organ processing (that is, facility, reagents, and procedures for receipt, inspection, and storage; tissue culture components, biological risk factors, and processing area), (2) donors (human and nonhuman) and screening, and (3) cell, tissue, and organ characterization and processing.
1.2 This guide does not apply to any medical products of human origin regulated by the U.S. Food and Drug Administration (FDA) under 21 CFR Parts 16 and 1270 (1) and 21 CFR Parts 207, 807, and 1271 (2).
1.3 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.
WITHDRAWN RATIONALE
This guide described the processing, characterization, production, and quality assurance of cells, tissues, and organs used for Tissue Engineered Medical Products (TEMPs).
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this guide was withdrawn in September 2015. This standard is being withdrawn without replacement due to its limited use by industry.

SIGNIFICANCE AND USE
The preservation of TEMPs can affect their subsequent characteristics (for example, structural, mechanical, biological, and metabolic properties). The aspects of preservation that can most adversely affect these characteristics include, but are not limited to, cooling, cooling rates, warming, warming rates, freezing, freezing rates, thawing, thawing rates, preservation medium, and pre- and post-processing.
The intent of this guide is to outline procedures that can minimize the loss or degradation of the desired characteristics of the TEMPs.
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1.1 This guide covers the development of standards related to the preservation of cells, tissues, and tissue engineered medical products (TEMPs). Preservation techniques include freezing, vitrification, and hypothermic preservation. This guide discusses preservation, including issues of pre-preservation processing, the process of preservation, storage, transport, recovery, post-preservation processing, quality assurance, and process control.
1.2 This guide contains general guidelines for the preservation of cells, tissues, and tissue engineered medical products (TEMPs) and will identify more specific parameters relevant to the preservation of TEMPs.
1.3 This guide does not apply to any medical products of human origin regulated by the U.S. Food and Drug Administration (FDA) under 21 CFR Parts 16 and 1270 and 21 CFR Parts 207, 807, and 1271.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory requirements prior to use.
WITHDRAWN RATIONALE
This guide covers the development of standards related to the preservation of cells, tissues, and tissue engineered medical products (TEMPs). Preservation techniques include freezing, vitrification, and hypothermic preservation. This guide discusses preservation, including issues of pre-preservation processing, the process of preservation, storage, transport, recovery, post-preservation processing, quality assurance, and process control.
Formerly under the jurisdiction of Committee F04 on Medical and Surgical Materials and Devices, this guide was withdrawn in January 2013 in accordance with section 10.5.3.1 of the Regulations Governing ASTM Technical Committees, which requires that standards shall be updated by the end of the eighth year since the last approval date.